1. Field of the Invention
The present invention relates to a composition for detection of HCV by a single step reaction, comprising a specific primer and probe. In particular, the present invention relates to a composition comprising the primer sequences of SEQ ID NOs: 1 and 2 for detection of HCV by a single step reaction, and a method for detecting HCV in blood using a composition comprising the probe of SEQ ID NO:5 or SEQ ID NO:9 for detection of HCV by a single step reaction.
2. Background Art
Hepatitis C virus (HCV), which is a kind of hepatitis virus, is a principal factor in causing serious diseases such as hepatitis including acute hepatitis and chronic hepatitis, which can develop into hepatic cirrhosis and hepatoma. HCV is transmitted via blood transfusion and fluid. It is estimated that about 4 hundred million people over the world are infected with the virus: 0.2-2% of people in the developed countries of Europe, North America and Japan; 2-5% in South America and Asia; over 5% in Africa; and 1.6% (8 hundred thousand) in Korea. See Park et al., J. Viral Hepat. 2:195-202 (1995). Unlike hepatitis B virus (HBV), HCV is a virus very threatening to human health in that 50-85% of people infected with hepatitis C virus eventually develop chronic hepatitis. Because HCV is an RNA virus, scientists have not developed any proper remedies and vaccines, much less basic study on HCV yet.
HCV is a positive-strand RNA virus consisting of about 9,500 bases and about 3,000 amino acids. HCV basically consists of an open reading frame (ORF) producing three structural proteins and six non-structural proteins.
Since the HCV level in blood is very low and HCV is an RNA virus, its infection cannot be detected by antigen assays, and thus antibody assays are usually performed. However, the antibody assays are problematic in that the antibodies cannot be detected for a latency period of 6 months after HCV infection. Furthermore, they are not detected in 10% of infected people, which increases the risk of HCV-positive blood transfusion.
On account of the low accuracy of HCV antibody assays, RT-PCR (reverse transcription-polymerase chain reaction) is used for direct detection of HCV RNA. However, there are disadvantages in that the procedures of separately performing reverse transcription and PCR are complicated so as to require skilled persons and much time, and the simultaneous use of two enzymes as in the previous method reduces specificity and sensitivity, and the accuracy of quantitative determination.
Nucleic acid amplification techniques including PCR, which is used to analyze the presence of specific nucleic acids or genes, requires a process of extracting DNA from a sample. The extraction procedures lead to an inevitable loss of DNA. For example, even though a sufficient amount of the target DNA is contained in the sample as a template for amplification, the suitable amount of DNA for amplification could not be recovered due to the loss during the extraction process, leading to a reduction in the amplification efficiency of the template. Therefore, it is hard to sufficiently amplify the target DNA and detect the target DNA in the sample. Consequently, the technique produces a false negative result, and thus it is difficult to ensure accurate results. Even though using the same sample, variations in DNA yields during the extraction process can produce different results, leading to the lack of reproducibility. That is, the DNA amplification technique has problems in that it requires the DNA extraction process to cause the loss of trace target DNA in the sample, whereby the detection sensitivity is reduced. In addition, when amplification inhibitors (e.g., heparin, surfactants, protein denaturants, organic solvents, etc.) are contained in the sample liquid, the amplification efficiency is also reduced lowering the detection sensitivity.